CY1205A - Penicillin derivatives - Google Patents

Description

GB 2 044 255 A

SPECIFICATION Chemical compounds

5 The present invention relates to hitherto unknown /3-lactam compounds including their salts with pharmaceutically acceptable, non-toxic acids or bases, to methods for producing said new compounds, to pharmaceutical compositions containing 10 the new compounds, to dosage units of the compositions, and to methods of treating patients suffering from infectious diseases using said new compounds.

The present invention provides new compounds 15 useful in the treatment of bacterial infections. The new compounds are in particular strongly active against ^-lactamase producing bacteria.

The compounds of the invention, which are valuable antibiotics in the human and veterinary prac-20 tice, are represented by the general formula i:

R-|—CH—CO—NH4 25 2 0-

3 I

in which Ri stands for a phenyl, 4-hydroxyphenyl, 30 1,4-cyclohexadienyl or a 3-thienyl group; R2 represents a primary amino or a carboxy group; R3 is a hydrogen atom, or a lower alkyl, aryl oraralkyl radical, preferably a methyl, phenyl or benzyl group, and A stands for a radical of a ^-lactamase inhibitor con-35 taining a /3-lactam ring as well as a carboxy group, A being connected via the carboxy group. More specifically, A is represented by one of the general formulae II, III, or IV:

40

45

50

55

60

in which R„ stands for a hydrogen or a halogen atom; R5 is a hydrogen atom or an amino or acylamino group, but at least one of R4 and R5 being hydrogen; R6 represents a halogen atom; and R7 65 stands for a hydroxyl group, or one of the radicals of known clavulanic acid derivatives with /3-lactamase inhibitory activity.

Generally, "lower alkyl" stands for a C-1 to C-6 straight or branched alkyl radical, aryl stands for a 70 monocyclic or bicyclic, carbocyclic radical, and acylamino stands for a radical present in the side chain of well-known penicillins. The asterisk in the side chain and, in case R3 is different from hydrogen, the dagger in the ester moiety indicate chiral centers 75 which give rise to diastereomeric forms of the compounds of formula I. The invention comprises ail such diastereomers as well as mixtures thereof.

The salts of the new compounds are salts with pharmaceutically acceptable, non-toxic acids or 80 bases, depending on whether R2 stands for a primary amino group or for a carboxy group.

Among suitable acids can be mentioned hydrochloric acid, hydrobromic acid, hydroiodic acid, phosphoric acid, sulphuric acid, nitric acid, 85 p-toluenesuiphonic acid, methanesulphonic acid, formic acid, acetic acid, propionic acid, citric acid, tartaric acid, maleic acid, pamoic acid, and p-(dipropylsulfamyl)benzoicacid (probenecid). Among suitable basic salts can be mentioned alkali 90 metal salts or alkaline earth metals salts, such as sodium, potassium, magnesium, or calcium salts as well as salts with ammonia or suitable non-toxic amines, such as lower alkylamines, e.g. triethylamine, hydroxy-lower alkylamines, for 95 example 2-hydroxyethylamine, bis-(2-

hydroxyethyl)-amine ortris - (2 - hydroxyethyl) -amine, cycloalkylamines, for example dicyclohex-ylamine, or benzylamines, for example N,N'-dibenzylethylenediamine or dibenzylamine, without 100 these examples being limiting the invention. Also salts with acidic or basic antibiotics are within the scope of the invention. In some instances, it is preferred to use easily soluble salts, whereas for other purposes, it may be appropriate to use an only 105 slightly soluble salt, e.g. in order to obtain a prolonged effect. In particular, a prolonged effect can be obtained by using a salt with probenecid which blocks the tubular excretion of/3-Iactam compounds.

In the clinical treatment of bacterial infections it is 110 a serious problem that/3-lactamase producing bacteria are occurring with increasing frequency. These enzymes inactivate most penicillins and cephalosporins, and it is well recognized that ^-lactamases from both gram-positive and gram-negative bacteria con-115 tribute significantly to the resistance of bacteria to /3-lactam antibiotics.

Several naturally occurring /3-lactamase inhibitors, including clavulanic acid and the olivanic acids, have been described. More recently, a number of semi-120 synthetic/3-lactam compounds, e.g. pen'icillanic acid 1,1-dioxide, 6a-chloropenicillanic acid 1,1-dioxide, a series of clavulanic acid derivatives, 6/3-bromopenicillanic acid, methicillin sulphone, and quinacillin sulphone, were found to possess similar 125 biological properties. With a few exceptions, these compounds display only weak antibacterial activity against most gram-positive and gram-negative organisms, but are powerful inhibitors of a wide range of/3 - lactamases. In combination with 130 selected penicillins and cephalosporins, the comBNSDOCID: <GB 2044255A_L>

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pounds act synergistically against a variety of /3-lactamase producing bacteria because they protect the penicillins and cephalosporins against inac-tivation.

5 As mentioned above, the present invention provides new compounds in particular intended for eternal use and being strongly antibacterially active in vivo. The advantageous effect against /3-lactamase producing bacteria is achieved because 10 the compounds contain in one and the same molecule both the moiety of an antibacterially highly active penicillin and the moiety of a potent /3-lactamase inhibitor. However, two prerequisites are necessary to utilize this feature of the new com-15 pounds. They must be capable of being absorbed from the gastro-intestinal tract, and during or after the absorption they must be hydrolyzed with liberation of the penicillin and the /3-lactamase inhibitor. It has turned out that both of these prerequisites are 20 fulfilled, and therefore the present compounds are valuable pro-drugs of both the penicillins and the /3-lactamase inhibitors.

thus, studies in animals and human volunteers have shown that the new compounds are readily 25 absorbed from the gastro-intestinal tract. During or afterthe absorption they are hydrolyzed with liberation of equimolar amounts of the two components in question, the penicillin and the /3-lactamase inhibitor, giving rise to simultaneous high blood and 30 tissue levels of the two components. Thereby the penicillins are protected against inactivation by the /3-lactamases.

The efficient absorption and in vivo hydrolysis of the compounds of the invention are illustrated by a 35 study in human volunteers dosed orally with one of the new compounds, namely the hydrochloride of 1,1-dioxopenicillanoyloxymethyl 6 - (D - a - amino - a - phenylacetamidojpenicillanate, in the following called VD-1827. For comparison, the same group of 40 volunteers was also given equimolar amounts of the orally active ampicillin pro-drug, pivampicillin, and potassium penicillanate 1,1-dioxide, respectively. The results of these studies are summarized in Tables I and II.

Table I

Serum concentrations and urinary excretion of ampicillin in fasting volunteers following oral administration of

A. 125 mg of pivampicillin free base in tablets.

B. 170 mg of VD-1827*1 hydrochloride (corresponding to 125 mg of pivampicillin free base) in aqueous solution.

Subject

Serum concentrations

1

H

Urinary excretion (£ of dose)

Hours after administration

0.25

0.

5

1

2

4

0-

6

0

'2k

A

B

A

£

A

B

A

B

A

B

A

B

A

B

GK

0.22

1.6

2.2

2.1

3.7

1.8

1.2

0.48

0.13

0.09

78

57

79

58

MK

<0.03

2.7

1.1

3.3

3-7

1.7

1.5

0.52

0.17

0.0 6

76

60

76

61

FJ

0.13

1.2

2.3

3.3

2.3

2.4

1.1

0.52

O.X7

0.0?

50

65

52

66

MM

0.?4

1.9

3.1

2.9

3.7

2.3

1-7

O.63

0,27

0.13

54

NS^

57

LA.

1.7

1.4

3.1

k„0

3*4

2.6

1.2

1.0

0.23

0.25

64

66

66

70

Mean

(0.48)

1.76

2.36

3.12

3.36

2.16

1.3k

0.63

0.19

0.12

67

60

68

62

VD-A827 a a 1,1-dioxopenicillanoyloxymi No sample

Table H

45 Urinary excretion in 0 to 6 hours of penicillanic acid 1,1-dioxide in fasting volunteers following oral administration of

A. 73 mg of potassium penicillanate 1,1-dioxide (corresponding to 63 mg of penicillanic acid

50 1,1 -dioxide) in aqueous solution

B. 170 mg of VD-1827 hydrochloride (corresponding to 63 mg of penicillanic acid 1,1-dioxide) in aqueous solution

Subject

Urinary excretion (c/0 of dose)

A

B

GK

2.5

60

MK

4.0

76

PJ

9.5

77

MM

5-5

63

LA

4.5

79

Msan

5.2

71

thyl 6-(D-a-amino-cc—phenylacetaraido) penicillanate

It will appearfrom Table I that oral administration 55 of VD-1827 gives rise to similar serum levels of ampicillin as obtained after an equimolar dose of pivampicillin. It also appears from Table I that the urinary recovery of ampicillin after administration of VD-1827 is comparable to that following administra-60 tion of pivampicillin.

As indicated in Table II, only 5.2% of penicillanic acid 1,1 -dioxide were excreted in the urine after oral administration of the corresponding potassium salt. In contrast thereto administration of an equimolar 65 amount of VD-1827 gave a 71% urinary recovery of penicillanic acid 1,1-dioxide, thus again illustrating the efficient oral absorption of VD-1827.

By using the compounds of the invention the antibacterial spectrum of the penicillin in question is 70 widely extended, as also /3-lactamase producing strains will be susceptible to treatment. As mentioned above, such /3-lactamase producing strains are found with increasing frequency and are a serious problem in the clinical therapy. The compounds

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GB 2 044 255 A 3

of the invention will for such purpose be of extreme value.

Therapeutically the new compounds have distinct advantages over mere combinations of the penicil-5 lins and the /3-lactamase inhibitors to which they are hydrolyzed, or combinations of orally active esters thereof.

For example, many of the/3-lactamase inhibitors, including penicillanic acid 1,1-dioxide, are absorbed 10 poorly or irregularly from the gastro-intestinal tract (cf. Table II). Also, many of the penicillins, including ampicillin and carbenicillin, are incompletely absorbed. In addition, individual variations in the rate of absorption of the various penicillins and .15 /3-lactamase inhibitors may in many instances lead to a situation where the active components are not present simultaneously or in the optimum ratio,

even if the two drugs are given simultaneously.

Certain easily hydrolyzable esters of penicillins 20 and /3-lactamase inhibitors are absorbed betterfrom the gastro-intestinal tract than the corresponding free acids. However, hydrolysis of such esters in the organism gives rise to the formation of inactive by-products, and although these by-products are 25 relatively non-toxic, it is undesirable to expose the organism to unnecessary metabolites. Another disadvantage by using combinations of easily hydrolyzable esters of the penicillins and the /3-lactamase inhibitors is that the ester moieties 30 increase the molecular weight of the compounds and consequently the size of the dosage u nit. By using the compounds of the invention, the size of the dosage units can be decreased considerably.

In addition, the absorption of such esters will nor-35 mally nottake place simultaneously, even if the compounds are given to the patient at the same time. For instance, the pivaloyloxymethyl ester of ampicillin is being absorbed very rapidly, whereas the sparingly soluble pivaloyloxymethyl ester of the 40 /J-lactamase inhibitor penicillanic acid 1,1-dioxide is being absorbed much more slowly.

All of these disadvantages are avoided by using the compounds of the invention.

It has been found that the in vitro synergy between 45 the different/3-lactamase inhibitors and various penicillins is particularly pronounced when the ratio between the two components is between 3:1 and 1:3. As the various penicillins have slightly different biological half-lives and distribution characteristics, 50 the ratio between the liberated components of the new compounds in the organs and tissues may vary to some degree, but will normally be within the above preferred limits.

The invention also comprises methods forthe 55 preparation of the compounds described above.

According to one method of the invention a compound of formula V:

60 Rj-CH—CO—HHj L-S

65

in which R,, R3, and A are as defined above, B stands for an azido group, a protected amino group, e.g. a benzyloxycarbonylamino.triphenylmethylamino, 1-methoxycarbonylpropen -2 -yl - amino or 1 -N,N -70 dimethylaminocarbonylpropen - 2 - yl - amino group, or a protected carboxy group, such as a ben-zyloxycarbonyl orcyanomethoxycarbonyl group, or similar known protected amino or carboxy groups, is subjected to a hydrogenolysis or hydrolysis depend-75 ing on what A and B stand for.

The reactions are performed in mixtures consisting of a suitable organic solvent, e.g. ethyl acetate or tetrahydrofurane, and water, in a ratio of3:1 to 1:3, preferably 1:1, and at temperatures from 0° to 30°C. 80 If B is an azido group or another group which can be converted into an amino or carboxy group by hydrogenolysis, e.g. palladium on carbon may be used as a catalyst, and if B is a group susceptible to hydrolysis, this may be catalyzed by acid, e.g. hydroch-85 loric, hydrobromic or sulphuric acid or p-toluenesulphonic acid.

The intermediates of formula V may be prepared by reacting a compound of formula VI:

9° - R|—CH—CO—I/S-

95

t—0—CH—X

R

3 H

in which R„ R3, and B have the meanings as defined above, and X stands for a leaving group, such as a halogen atom, with a compound of formula A-M in 100 which A is as defined before and M is a cation, such as Na+, K+, an ammonium ion, a tri- or a tetraal-kylammonium ion, e.g. a tetrabutylammonium ion.

The reaction is performed in a suitable solvent, e.g. dimethylformamide, ethyl acetate, dich-105 loromethane, acetone or hexamethyl phosphoric acid triamide, for a sufficient time and at an adequate temperature with a view to accomplish the desired conversion, usually at a temperature from 0° to 60°C.

110 Another method for the preparation ofthe intermediates of formula V comprises a first step in which a compound A-M is reacted with a compound of formula VII to afford an intermediate of formula VIII:

115

Y-CH-X

I

R_

A-CH-X

'3 I

in which formulae R3, and X are as defined above, 120 and Y is a bromine or iodine atom, an alkylsul-

phonyloxy, arylsulphonyloxy, chlorosulphonyloxy, or a - haloalkoxysulphonyloxy radical, Y being a better leaving group than X.

The reaction is performed in the same manner as 125 described forthe preparation ofthe known compounds of formula VI and takes place in a suitable solvent, e.g. dimethylformamide, ethyl acetate, dich-loromethane, acetone or hexamethyl phosphoric acid triamide, usually at a temperature from 0°to 130 60°C.

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GB 2 044 255 A

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In a second step the intermediate of formula VIII is reacted with a penicillin derivative of formula IX:

R-j—CH—CO—NH^i U

B

--C—0—M

0=

3 X

mulae V, VIII and XI are unknown compounds and are also within the scope ofthe present invention.

A further embodiment ofthe method, by which the compounds of formula I, R2 being a primary amino 70 group, can be prepared directly by a one-step procedure, comprises reacting a salt of an aminopencillin, e.g. ampicillin or amoxycillin, represented by the general formula XIII:

10 in which R„ B, and M are as defined above to form the intermediate of formula V. If desired, the X in formula VIII can in advance be exchanged by a better leaving group.

Another embodiment ofthe method comprises a 15 first step in which a compound of formula A-M is reacted with a 6-aminopenicillanic acid ester of formula X or an amino-protected derivative thereof, e.g. a trialkylsilyl derivative, to afford a compound of formula XI:

20 H H

)==I N

H X-O-^-X

Ri-CH-CO—! NH,

25

30

n=i M —-3^

H "-C—0—CH—A

\ I

0 R3 H

35 in which formulae R3, A, and X are as defined before. Hie reaction is performed in a suitable organic solvent, e.g. dimethylformamide, and at temperatures between 0° and 30°C.

Alternatively, the intermediates of formulaXI can 40 be prepared by reacting 6-aminopenicillanic acid or a salt or an amino-protected derivative thereof with a compound of formula VIII.

In a second step a compound of formula XI or a trialkylsilyl derivative thereof is reacted with a reac-45 tive derivative of an acid of formula XII:

R-,—CH—COOH

1 |

B JL

50 in which R, and B are as defined above. B can in addition be NH3+, Hal". The reactive derivative can for instance be an acid halide, such as an acid chloride or acid bromide; an acid anhydride; a mixed anhydride with an alkyl-carbonic acid, a car-55 boxylic acid, an inorganic acid or a sulphonic acid; or a radical obtained by reacting the free acid of formula XII with a carbodiimide or N,N'-carbonyl-diimidazole or a similarly functioning compound. The reaction can be performed in an organic solvent 60 or in a mixture thereof with water at low or slightly elevated temperature. Suitable solvents are dich-loromethane, chloroform, ethyl acetate, acetone, dimethylformamide, dimethylacetamide, ether, dioxane or other inert solvents.

65 The starting materials or intermediates of for-

75

80

with a compound of formula VIII, in which formulae R„ R3, M, A and X are as defined before, and preferably X stands for an iodine atom. The reaction is performed in a suitable organic solvent, e.g. ethyl 85 acetate, dichloromethane, chloroform, dimethylformamide, and at temperatures between 0° and40°C, preferably at room temperature.

The starting materials of formulae VI, VII, IX, and X are known or may be prepared by methods analog-90 ous to those used forthe preparation of similar known compounds.

Most ofthe starting materials of formula A-M or the corresponding acids are known compounds. New compounds are acids and salts corresponding 95 to A being a radical of formula II in which Rs stands for certain acylamino radicals. The latter compounds are penicillin sulphones, which may be prepared by known methods.

The compounds of formula I can be purified and 100 isolated in usual manner and may be obtained either as such or in the form of a salt.

The compounds may in some cases be obtained as diastereomeric mixtures which when desired may be separated by known methods, e.g. chromatogra-105 phy.

It is a further object of the present invention to provide pharmaceutical compositions which are useful in the treatment of infectious diseases in the human and veterinary practice, and which may be 110 used for enteral, parenteral or topical administration.

With this object in view, the compositions ofthe invention contain as an active component at least one member selected from the group consisting of compounds ofthe formula I and salts thereof as 115 defined above, together with solid or liquid pharmaceutical carriers and/or diluents.

In the said compositions, the proportion of therapeutically active material to carrier substance can vary between 1% and 95% by weight. The com-120 positions can be worked up to various pharmaceutical forms of presentation, such as tablets, pills, dragees, suppositories, capsules, sustained-release tablets, suspensions and the like containing the compounds of formula 1 or their atoxic salts, as 125 defined above, mixed with carriers and/or diluents.

Pharmaceutically acceptable, non-toxic, organic or inorganic, solid or liquid carriers and/or diluents can be used to make up compositions containing the present compounds. Gelatine, lactose, starch, mag-130 nesium stearate,talc, vegetable and animal fats and

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oils, gum, polyalkylene glycol, buffers or other known carriers, auxiliary agents and/or diluents for medicaments are all suitable.

Furthermore, the compositions may contain other 5 therapeutically active components which can appropriately be administered together with the present compounds in the treatment of infectious diseases, such as other antibacterials, antitussiva, pain-relieving drugs, probenecid, etc. In particular, 10 antibacterials, which act synergistically with one or both ofthe active components formed by in vivo hydrolysis ofthe compounds ofthe invention, are appropriate.

The compounds of formula I can be used either as 15 such or in the form of a salt. The compounds as such are only slightly soluble in water, whereas many of the salts, e.g. the hydrochlorides and the sodium salts, are readily soluble in water.

As indicated above, the present compounds may 20 be worked up to pharmaceutical forms of presentation including suspensions and non-aqueous ointments. A pharmaceutical preparation for oral treatment may be in the form of a suspension of one of the present compounds, the preparation containing 25 from 10 mg to 100 mg per ml ofthe vehicle.

Another object ofthe invention resides in the selection of a dose ofthe compounds ofthe invention and a dosage unit ofthe compositions ofthe invention which dose and dosage unit can be 30 administered so that the desired activity is achieved without simultaneous secondary effects. In the human therapy, the present compounds are conveniently administered (to adults) in dosage units of the compositions containing not less than 50 mg and 35 up to 2500 mg, preferably from 100 mg to 1000 mg calculated as the compound of formula I.

By the term "dosage unit" is meant a unitary, i.e. a single dose which is capable of being administered to a patient, and which may be readily handled and 40 packed, remaining as a physically stable unit dose comprising eitherthe active material as such or a mixture of it with solid or liquid pharmaceutical diluents, carriers, solvents and/or auxiliary agents.

In the form of a dosage unit, the compound may 45 be administered once or more times a day at appropriate intervals, always depending, however, on the condition ofthe patient, and in accordance with the prescription made by the medical practitioner.

50 Thus a daily dose will preferably be an amount of from 0.25 to 15 g of a compound of formula I or an equivalent amount of a salt thereof as defined before, which conveniently can be divided into sev-! era! single doses.

55 In the continuous therapy of patients suffering from infectious diseases, the tablets or capsules are the appropriate form of pharmaceutical preparation, if desired in the form of sustained-release formulations.

60 In the veterinary practice the above pharmaceutical compositions may also be used, preferably in the form of dosage units containing from 50 mg up to 25 g ofthe compound of formula I or a corresponding amount of a salt thereof.

65 Forthe treatment of mammary disorders, especially bovine mastitis, the antibacterial agent can be administered by the intramammary route in liquid or semiliquidform,such as an ointment, ortogether with a substantially water-insoluble and oil-insoluble 70 binding agent in the form of granules.

Still another object ofthe invention is to provide a method of treating patients suffering from infectious diseases, the method comprising administering to adult patients an effective amount of a compound of 75 formula I, either as such or in the form of a salt as defined before, and preferably, in the form ofthe dosage units aforesaid. The compounds of formula I are typically administered in amounts of 3 - 200 mg/kg body weight ofthe patient/day, correspond-80 ing to, for adult human patients, from 0.25 g to 15 g per day, or an equivalent amount of a salt as defined before of a compound of formula I.

In the treatment of patients, the present compounds can be administered either alone ortogether 85 with other therapeutically active compounds, e.g. probenecid, which aid in combatting the bacterial infection. Such combined treatment can be performed with formulations containing more or all of the therapeutically active compounds, or these may 90 be administered in separate formulations, these being given simultaneously or with suitable intervals.

In the treatment of patients, the daily dose is administered either at one time, or in divided dos-95 ages, e.g. two, three of four times a day.

In the following "Preparations" the methods for preparing new starting materials and intermediates are more specifically described.

Preparation 1 100 6a-Bromopenicillanic acid 1,1-dioxide

To a stirred solution of potassium permanganate (1.90 g, 12 mmol) in water (35 ml) and acetic acid (1.36 ml, 24 mmol) was added dropwise atO-5°C an icecold solution of potassium 6a-bromopenicil!anate 105 (1.91 g, 6 mmol) in water (25 ml). After the addition was finished (about 15 minutes), the mixture was stirred for another 20 minutes at the low temperature. The cooling-bath was removed, and to the mixture was added solid sodium pyrosulphite (1.52 g, 8 110 mmol) to reduce excess oxidation reagent. Precipitated manganese oxides were filtered off, and to the filtrate (about 60 ml) was added solid sodium chloride (20 g) and ethyl acetate (50 ml). The pH of the mixture was adjusted to 1.5 by addition of 4 N 115 hydrochloric acid with stirring, and the organic phase was separated. The aqueous phase was reex-tracted with ethyl acetate (25 ml), and the combined organic extracts were washed with saturated aqueous sodium chloride, dried, and evaporated//! 120 vacuo. The amorphous residue thus obtained was crystallized from ether-diisopropyl ether to afford 6a - bromopenicillanic acid 1,1-dioxide, melting point: 124-127°C.

A crystalline potassium salt ofthe above com-125 pound was obtained by addition of 1 M potassium 2-ethylhexanoate in acetone (3.6 ml) to a stirred solution of 6a-bromopenicillanic acid 1,1-dioxide (0.94 g, 3 mmol) in acetone (12 ml).

The NMR spectrum of potassium 6a -130 bromopenicillanate 1,1-dioxide (CD30D) showed

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signals at S = 1.48 (s, 3H; 2-CH3), 1.59 (s, 3H; 2-Ctf3), 4.48 (s, 1H; 3-H), 5.10 (d, J=2Hz, 1H; 6-H), and 5.35 (d, J=2Hz, 1H; 5-H) ppm. Tetramethylsilane was used as internal reference.

5 Preparation 2

6a-ChJoropenicillanic acid 1,1-dioxide

By substituting potassium 6a-chloropeniciIlanate forthe potassium 6a-bromopenicillanate in the procedure of Preparation 1,6a-chioropenicillanic acid 10 1,1-dioxide was obtained as crystals from diisop-ropyl ether, melting point: 134-137°C.

The NMR spectrum (CDCI3) showed signals at 8 = 1.50 (s,3H; 2-Ctf3), 1.64 (s, 3H; 2-CH3), 4.46 (s, 1H; 3-H), 4.70 (d, J= 1.5Hz, 1H; 6-H), and 5.18 (d, J=1.5Hz, 15 1H; 5-H) ppm. Tetramethylsilane was used as internal reference.

A crystallinepofass/c/n? salt ofthe above compound was obtained by addition of an equimolar amount of 0.8 M potassium 2-ethylhexanoate in 20 acetone to a stirred solution of 6a-chtoropenicillanic acid 1,1-dioxide in acetone.

Preparation 3 Chioromethyl penicillanate 1,1-dioxide To a solution of penicillanic acid 1,1-dioxide (1.17 25 g, 5 mmol) in dimethylformamide (7.5 ml) was added triethylamine (0.98 ml, 7 mmol) and chloroiodomethane (2.18 ml, 30 mmol), and the mixture was stirred at room temperature for4 hours. After dilution with ethyl acetate (30 ml), the mixture 30 was washed with water (3 x 10 ml) followed by saturated aqueous sodium chloride (5 ml), dried, and evaporated in vacuo to leave the desired compound as a yellowish oil, which crystallized from ether-petroleum ether, melting point: 94-96°C. 35 The NMR spectrum (CDCU showed signals at8 =

1.47 (s,3H; 2-Ctf3), 1.66 (s,3H; 2-CW3),3.53 (d, J=3Hz, 2H; 6a-H and 6/3-//), 4.46 (s,1H; 3-W),4.68 (t, J=3Hz,

1H; 5-H), and 5.85 (ABq, J=6Hz, 2H; OCW2CI) ppm. Tetramethylsilane was used as internal reference. 40 Preparation 4

1-Chioroethyl penicillanate 1,1-dioxide

Following the procedure of Preparation 3, but substituting 1-chloro-1-iodoethane forthe chloroiodomethane and increasing the reaction time 45 to 16 hours, crude 1-chloroethyl penicillanate 1,1-dioxide was obtained as a yellow oil which could be purified by dry column chromatography on silica gel (ethyl acetate-petroleum ether, 7:3).

Preparation 5 50 Chioromethyl 6a-bromopeniciHanate 1,1 - dioxide By substituting 6a-bromopenicillanicacid 1,1-dioxide forthe penicillanic acid 1,1-dioxide in the procedure of Preparation 3, chioromethyl 6a -bromopenicillanate 1,1-dioxide was obtained as a 55 yellowish oil.

The NMR spectrum (CDCU showed signals atS =

1.48 (s, 3H; 2-CH3), 1.64 (s, 3H; 2-Ctf), 4.46 (s, 1H; 3-WK4.71 (d, J=1.5 Hz, 1H; 6-H), 5.17 (d, J=1.5 Hz, 1H; 5 -H). and 5.80 (ABq, J=6 Hz, 2H; OCW2CI) ppm.

60 TMS was used as internal reference.

Preparation 6 Chlorometh y! 6^-bromopenicillanate

By substituting potassium 6/3-bromopenicillanate forthe penicillanic acid 1,1-dioxide and the 65 triethylamine in the procedure of preparation 3,

BNSDOCID: <GB 2044255A_L>

chioromethyl 6/J-brompeniciIlanate was obtained as a viscous oil.

Preparation 7 Chioromethyl clavulanate 70 Following the procedure of Preparation 3, but substituting sodium clavulanate for the penicillanic acid 1,1-dioxide and the triethylamine, chioromethyl clavulanate was obtained.

Preparations 75 Chioromethyl penicillanate 1,1-dioxide

To a suspension of potassium penicillanate 1,1-dioxide (1.08 g) in dimethylformamide (12 ml) was added bis-chloromethyl sulphate (1.6 g), and the mixture was stirred at room temperature for45 80 minutes. After dilution with ethyl acetate (50 ml), the = mixture was washed with water followed by aqueous sodium bicarbonate, dried and evaporated in vacuo to leave an oil which was purified by chromatography on silica gel to yield the desired 85 compound, identical with the compound described in preparations.

Preparation 9 Chioromethyl 6a-chioropenicillanate 1,1-dioxide By substituting 6a-chloropenicillanic acid 1,1-90 dioxide forthe penicillanic acid 1,1-dioxide in the procedure of Preparation 3, chioromethyl 6a -chloropenicillanate 1,1-dioxide was obtained as a viscous oil.

The NMR spectrum (CDC13) showed signals at 8 = 95 1.48 (s, 3H; 2-CW3), 1.64 (s, 3H; 2-CW3),4.47 (s, 1H; 3-H), 4.68 (d, J=1.5 Hz, 1H; 6-H), 5.17 (d, J=1.5 Hz, 1H; 5-H), and 5.81 (ABq, J=6 Hz, 2H; 0CW2CI) ppm. TMS was used as internal reference.

Preparation 10 100 lodomethyipenicillanate 1,1-dioxide

To a solution of chioromethyl penicillanate 1,1-dioxide (5.6 g, 20 mmol) in acetone (45 ml) was added sodium idiode (9 g), and the mixture was stirred at room temperature for 16 hours. Precipitated 105 sodium chloride (1.15 g) was filtered off, the solvent was removed//? vacuo, and the residue thus obtained was treated with ethyl acetate-ether (1:1). Insoluble sodium iodide (6 g) was filtered off, and the filtrate was evaporated at reduced pressure. 110 The residue oil was purified by column chromatography on silica gel (ethyl acetate-n-hexan, 4:6) to yield the title compound as colourless crystals from ether, melting point: 101-102°C.

Preparation 11 115 6fi-Aminopenicillanic acid 1,1-dioxide hydrate A. 6(3-Benzyloxycarbonylaminopenicillanic acid 1,1-dioxide

To a stirred solution of 6/3 - benzyloxycar-bonylaminopenicillanic acid (63.5 g) and potassium * 120 hydrogen carbonate (18.1 g) in water (1125 ml) was slowly (about45 minutes) at0°C added a solution of potassium permanganate (38 g) in water (915 ml). During the oxidation, a pH of 6.5 was maintained in the reaction mixture by addition of dilute sulphuric 125 acid. Insoluble material was removed by filtration, and the filtrate was extracted with ethyl ether. The resulting aqueous phase was filtered again and, after addition of ethyl acetate (600 ml), acidified to pH 2.5 with stirring. The organic layer was separated, and 130 the aqueous phase was extracted with additional

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ethyl acetate (2 x 300 ml). After drying, the combined ethyl acetate extracts were evaporated//? vacuo. The residue was recrystallized from ethyl acetate (250 ml)-petroieum ether (500 ml) to yield the 5 pure compound, melting point:

153-154°C;[a]g°: +146.9° (c=1, 96% C2H5OH).

B. 6f3-Aminopenicillanic acid 1,1-dioxide hydrate Afiltered solution of 6/3 - benzyloxycar-10 bonylaminopenicillanic acid, 1,1-dioxide (15.3 g) and potassium hydrogen carbonate (4 g) in water (160 ml) was hydrogenated over 10% Pd/BaS04 (5 g) for4 hours at slightly elevated pressure. After filtration and extraction with ethyl ether (100 ml), the pH of the 15 ice-cold aqueous solution was adjusted to 2.5. The precipitate thus formed was filtered off, washed with water, and air-dried. Recrystallization from dimethylformamide-water afforded the pure mono-hydrate; melting point: 199-200°C

^ (dec.);[a]p°: +252.9°(c=1, dimethylformamide).

Preparation 12 Chioromethyl 1,1-dioxopeniciilanate To a mixture of potassium 1,1-dioxopenicillanate 25 (2.7 g, 10 mmol), potassium hydrogen carbonate (6.0 g, 60 mmol) and tetrabutylammonium hydrogen sulphate (0.34 g, 1 mmol) in water (10 ml) and dich-loromethane (15 ml), chioromethyl chlorosulphate (1.5 ml) was added. After stirring for 1 hour at30°C, 30 the mixture was filtered and the organic layer was separated and dried (sodium sulphate). After dilution with propanol-2 (25 ml), the solution was concentrated to about 10 ml in vacuo and left at 5°C for 1 hour. The crystals were filtered off, washed with cold 35 propanol-2 and dried in vacuo to give the title compound as colourless crystals with a melting point of 94-96°C.

Preparation 13 1-Chloroethy11,1-dioxopenicillanate 40 To a mixture of potassium 1,1-dioxopenicillanate (40.7 g, 0.15 mol), silver nitrate (25.5 g, 0.15 mol), and silver oxide (7.5 g) in acetonitrile (750 ml), 1-chloro-1-iodoethane (42 ml) was added. After stirring for 48 hours at ambient temperature, the silver 45 salts were filtered off, and the filtrate taken to dryness//? vacuo. The residue was dissolved in ethyl acetate (200 ml), and the solution was washed with saturated aqueous sodium chloride, filtered, dried, and evaporated//? vacuo. Chromatography ofthe 50 residue on silica gel (hexane-ethyl acetate, 3:2) gave the title compound as a crystalline mixture ofthe two diastereomers with m.p. 130-132°C.

Preparation 14 1-lodoethyl 1,1-dioxopeniciilanate 55 To a solution of 1-chloroethyl 1,1-

dioxopenicillanate (30 g, ~0.1 mol) in acetone (100 ml), sodium iodide (30 g, 0.2 mol) was added, and the mixture was stirred at ambient temperature for3 days. Aqueous sodium thiosulphate was added, and 60 the acetone was removed//? vacuo. The separated oil was dissolved in ethyl acetate, and the solution was washed with water, dried and evaporated in vacuo. The residual oil was chromatographed on silica gel (hexane-ethyl acetate, 3:1) to give a crystal-65 line mixture (m.p. 134-36°C) ofthe diastereomeric

1-iodoethyl and 1-chloroethyl esters, containing 40% ofthe iodo compound, according to the microanalyt-ical determination of iodine.

Preparation 15 70 Chioromethyl6fi-bromopeniciHanate To a stirred solution of potassium 6/3 -bromopenicillanate(0.96 g, 3 mmol) and potassium bicarbonate (1.80 g, 18 mmol) in water (9 ml) and ethyl acetate (9 ml) was added tetrabutylammonium 75 hydrogen sulphate (0.10 g, 0.3 mmol), followed by chioromethyl chlorosulphonate (0.45 ml, 4.5 mmol), and the mixture was stirred at room temperature for 1.5 hours. The organic phase was separated, and the aqueous phase re-extracted with ethyl acetate (9 ml). 80 The combined organic extracts were washed with water (2x5 ml), dried, and concentrated to about 5 ml at reduced pressure. The concentrate was subjected to dry column chromatography on silica gel (petroleum ether-ethyl acetate, 9:1) to afford pure 85 chioromethyl 6/3-bromopenicillanate as an almost colourless oil.

The NMR spectrum (CDCI3) showed signals at 8 = 1.54 (s, 3H; 2-CW3), 1.70 (s, 3H; 2-Ctf3),4.54 (s, 1H; 3-H), 5.35 and 5.59 (2d, J=4Hz, 2H; 5-H and 6-H), and 90 5.77 (ABq, J=5Hz, 2H; OCW2CI) ppm. Tetramethylsilane was used ac internal reference.

Preparation 16 lodomethyi 6fi-bromopenicillanate To a solution of chioromethyl 6/3 - bromopenicilla-95 nate (0.82 g, 2.5 mmol) in acetone (5 ml) was added solid sodium iodide (0.75 g, 5.0 mmol), and, after protection from light, the mixture was stirred at room temperature for 24 hours. Precipitated sodium chloride was filtered off, washed with acetone (2x1 100 ml), and the filtrate was evaporated in vacuo to leave an oily residue which was re-dissolved in ethyl acetate (20 ml). The resulting solution was washed with water (2x10 ml), dried (MgS04), and, following concentration to about 5 ml at reduced pressure, sub-105 jected to column chromatography on silica gel using petroleum ether-ethyl acetate, 9:1, as the eluent. Fractions containing the pure title compound, as revealed by thin-layer chromatography (TLC), were combined and evaporated//? vacuo to yield 110 iodomethyl 6/3 - bromopenicillanate as a slightly yellowish oil.

The NMR spectrum showed signals at 8 = 1.55 (s, 3H; 2-CH3), 1.69 (s, 3H; 2-CH3), 4.50 (s, 1H; 3-H). 5.34 and 5.57 (2d, J=4Hz, 2H; 5-H and 6-H), and 5.97 115 (ABq, J=5Hz,2H; OCtf2l) ppm. Tetramethylsilane was used as internal reference.

Preparation 17 Chioromethyl 1,1 - dioxo -6/3 -(2, 6- dimethoxyben-zamidojpeniciflanate 120 Chioromethyl chlorosulphate (1.8 ml, 18 mmol) was added during 20 minutes at room temperature to a mixture of 1,1-dioxo - 6/3 - (2,6 - dimethoxyben-zamido)penicillanicacid (methicillin sulphone; 6.2 g, 15 mmol), potassium hydrogen carbonate (8.7 g, 87 125 mmol) and tetrabutylammonium hydrogen sulphate (0.51 g, 1.5 mmol) in water (15 ml) and dich-loromethane (15 ml).

After stirring for a further 15 minutes, the organic phase was separated, dried, and evaporated in 130 vacuo to leave an oil which crystallized from 96%

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ethanol to yield colourless crystals with m.p. 142-143°C (dec). Two recrystallizations from acetone-water gave the analytical sample with m.p.

154-155°C (dec) ;[a] : +195°(c=1,CHCi3).

Preparation 18 lodomethyl 1,1- dioxo -6(3- (2,6- dimethoxyben-zamido)per>icillanate

Sodium iodide (3 g, 20 mmol) was added to a solu-10 tion of chioromethyl 1,1-dioxo - 6/3 - (2,6 - dimethox-ybenzamido)penicilianate (2.31 g, 5 mmol) in acetone (10 ml), and the mixture was stirred overnight at room temperature. Addition of water precipitated the title compound as crystals which were 15 collected byfiltration and dried in vacuo; m.p. 153-156°C (dec).

The product was dissolved in a mixture of acetone and 96% ethanol, the acetone was removed in vacuo and the desired compound crystallized. By repeating 20 this procedure the m.p. was raised to 169-170°C

(dec);[a]2°: +197° (c=1, CHCI3).

Preparation 19 Chioromethyl 1,1-dioxo-6a-chioropenicillanate 25 By substituting potassium 1,1-dioxo-6a-chloropenicillanate forthe potassium 6/3 -bromopenicillanate in the procedure of Preparation

15, the title compound was obtained as colourless crystals from ether-diisopropyl ether; melting point:

30 111-113°C; [a]p°: +210° (c=0.5, CHCI3).

Preparation 20 lodomethyl J, 1-dioxo-6a-chloropenicillanate By substituting chioromethyl 1,1 - dioxo -6a-35 chloropenicillanateforthe chioromethyl 6/3-

bromopenicillanate in the procedure of Preparation

16, the title compound was obtained as a colourless foam.

The NMR spectrum (CDCI3) showed signals at8 = 40 1.49 (s, 3H; 2-CW3), 1.62 (s, 3H; 2-CH3), 4.41 (s, 1H; 3-H). 4.66 and 5.16 (2d, J=1.S Hz, 2H; 5-H and 6-H), and 6.01 (ABq, J=5 Hz, 2H; OCW2l) ppm. Tetramethylsilane was used as internal reference. Preparation 21 45 Chioromethyl 1,1-dioxo-6a-bromopeniciilanate By substituting potassium 1,1 - dioxo - 6a -bromopenicillanate forthe potassium 6/3-bromopenicillanate in the procedure of Preparation

15, the title compound was obtained as colourless 50 crystals from ether-diisopropyl ether; melting point:

92-93°C; [a]p°:+ 185° (c=0.5, CHC13).

Preparation 22 55 lodomethyl 1,1 -dioxo-6a-bromopeniciHanate By substituting chioromethyl 1,1- dioxo - 6a -bromopenicillanate for the chioromethyl 6/8 -bromopenicillanate in the procedure of Preparation

16, the title compound was obtained as a colourless 60 foam which failed to crystallize.

The NMR spectrum (CDCI3) showed signals at 8 = 1.49 (s, 3H; 2-CW3), 1.63 (s, 3H; 2-CW3), 4.41 (s, 1H; 3-H), 4.70 and 5.16 (2d, J=1.5 Hz, 2H; 5-H and 6-H), and 6.01 (ABq, J=5 Hz, 2H; OCW2l) ppm. Tet-65 ramethyfsilane was used as internal reference.

Preparation 23 Chioromethyl 6f3-iodopenicilianate

By substituting potassium 6/3-iodopenicillanate for the potassium 6/3-bromopenicillanate in the proce-70 dure of Preparation 15, the title compound was obtained as a slightly yellowish oil.

The NMR spectrum (CDCI3) showed signals at 8 =

1.52 (s,3H; 2-CW3), 1.71 (s,3H; 2-CW3),4.55 (s, 1H; 3-H), 5.40 and 5.63 (2d, J =3.5 Hz, 2H; 5-H and 6-H),

75 and 5.78 (ABq, J=5.5 Hz, 2H; 0CW2CI) ppm. Tetramethylsilane was used as internal reference.

Preparation 24 lodomethyl 6/3-iodopenicillanate By substituting chioromethyl 6/3-iodopenicillanate 80 forthe chioromethyl 6/3-bromopenicillanate in the procedure of Preparation 16, the title compound was obtained as a yellowish oil.

The NMR spectrum (CDCI3) showed signals at 8 =

1.53 (s,3H; 2-CW3), 1.70 (s,3H; 2-CH3). 4.53 (s, 1H; 85 3-H), 5.39 and 5.61 (2d, J=3.5 Hz, 2H; 5-H and 6-H),

and 6.00 (ABq, J=5.5 Hz, 2H; 0CW2I) ppm. Tetramethylsilane was used as internal reference.

Preparation 25 Chioromethyl 6/3-chloropenicillanate 90 By substituting potassium 6/3-chloropenicillanate forthe potassium 6/3-bromopenicillanate in the pro-cedu re of Preparation 15, the title compound was obtained as a colourless oil.

The NMR spectrum (CDC!3) showed signals at 8 = 95 1.53 (s, 3H; 2-CW3), 1.69 (s, 3H; 2-CW3), 4.54 (s, 1H; 3-H), 5.24 and 5.62 (2d, J=4 Hz, 2H; 5-H and 6-H), and 5.80 (ABq, J=5 Hz, 2H; 0CH2CI) ppm. Tetramethylsilane was used as internal reference.

Preparation 26 100 lodomethyl 6f3-chloropenicillanate

By substituting chioromethyl 6/3 - chloropenicillanate forthe chioromethyl 6/3 - bromopenicillanate in the procedure of Preparation 16, the title compound was obtained as a slightly yellowish oil.

105 The NMR spectrum (CDCI3) showed signals at 8 = 1.52 (s, 3H; 2-CH3), 1.69 (s, 3H; 2-CH3), 4.52 (s, 1H; 3-H), 5.22 and 5.58 (2d, J=4 Hz, 2H; 5-H and 6-H), and 5.99 (ABq, J=5 Hz, 2H; 0CW2l) ppm. Tetramethylsilane was used as internal reference. 110 Preparation 27

Ch/orometh yl 6/3-bromopenicillanate

A. Chioromethyl 6,6-dibromopeniciHanate By substituting potassium 6,6-

dibromopenicillanate forthe potassium 6/3 -115 bromopenicillanate in the procedure of Preparation 15, the title compound was obtained as a slightly yellowish oil which crystallized from ether-diisopropyl ether; melting point:

120 105"1 °7C: +206° (c=0-5< CHCIj).

The NMR spectrum (CDCI3) showed signals at 8 =

1.54 (s, 3H; 2-CW3), 1.66 (s, 3H; 2-CH3), 4.60 (s, 1H; 3-H), 5.80 (ABq, J=5 Hz, 2H; OCW2CI), and 5.83 (s, 1H; 5-H) ppm. Tetramethylsilane was used as internal

125 reference.

B. Chioromethyl6f3-bromopenicillanate To a stirred solution of chioromethyl 6,6-

dibromopenicillanate (1.63 g, 4 mmol) in dry benzene (40 ml) was added under nitrogen at 0°C tri-/j-130 butyltin hydride (1.16 g, 4 mmol). After stirring at

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room temperature for 18 hours, the mixture was evaporated in vacuo. The residual oil was purified by dry column chromatography on silica gel (petroleum ether-ethyl acetate, 85:15) to yield pure 5 chioromethyl 6/J-bromopenicillanate as a slightly yellowish oil.

The NMR spectrum ofthe product was identical with that of the compound described in Preparation 15.

10 Preparation 28

Bromomethyl 1,1-dioxopeniciilanate

To a solution of sodium bromide (1.0 g) in N,N-dimethylformamide (10 ml) was added chioromethyl 1,1-dioxopenicillanate (0.28 g, 1 mmol), and the mix-15 ture was stirred at room temperature for 20 hours. After dilution with ethyl acetate (50 ml), the mixture was washed with water (4x10 ml), dried, and evaporated in vacuo. The residue was purified by column ; chromatography on silica gel to yield the desired 20 compound as a yellowish oil.

The NMR spectrum (CDCI3) showed signals atS = 1.49 (s, 3H; 2-CH3), 1.64 (s, 3H; 2-CH3), 3.52 (m, 2H; 6-H, 4.47 (s, 1H; 3-H), 4.75 (m, 1H; 5-H), and 5.98 (ABq, J =4.5 Hz, 2H; 0CW2Br) ppm. TMS was used as 25 interna! reference.

The invention will be further described in the following Examples which are not to be construed as limiting the invention.

Example 1

30 1,1 - DioxopeniciHanoyioxymethyi 6-(D-a- amino -a - phenylacetamidojpenicillanate hydrochloride A. 1,1 - DioxopeniciHanoyioxymethyi 6-(D-a-azido -a-phenylacetamidojpenicillanate

To a solution of chioromethyl 6 - (D - a - azido - a -35 phenylacetamidojpenicillanate (2.54 g, 6 mmol) in dimethylformamide (35 ml) was added potassium penicillanate 1,1-dioxide (1.63 g, 6 mmol), and the mixture was stirred at room temperature for 20 hours. After dilution with ethyl acetate (140 ml), the 40 mixture was washed with water (4 x 35 ml), followed by saturated aqueous sodium chloride (20 ml), and the organic phose was dried and evaporated in vacuo. The yellow oily residue thus obtained was purified by dry column chromatography on 45 silica gel (cyclohexane-ethyl acetate, 1:1) to yield the desired compound as a yellowish oil.

The NMR spectrum (CDCI3) showed signals at 8 = 1.43 (s, 3H; 2-CH3), 1.52 (s, 3H; 2-CW3), 1.59 (s, 3H; 2-CH3), 1.66 (s, 3H; 2-CW3), 3.48 (d, J=3Hz, 2H; 6 a-H 50 and6/3-H),4.44 (s, 1H; 3-H),4.51 (s, 1H; 3-//),4.63 (t, J=3Hz, 1H; 5-H), 5.13 (s, 1H; CHN3), 5.65 (m,2H; 5-H and 6-H), 5.92 (s, 2H; 0CW20), and 7.48 (s, 5H; arom. CH) ppm. Tetramethylsilane was used as internal ' reference.

55 B. 1,1-Dioxopenicillanoyloxymethyl 6-(D-a-amino - a-phenylacetamidojpenicillanate hydrochloride

A solution of 1,1 -dioxopenicillanoyloxymethyl 6 -(D - a - azido - a - phenylacetamidojpenicillanate 60 (1.77 g, 2.85 mmol) in ethyl acetate (25 ml) was placed in a three-necked flask, equipped with a gas in let/outlet tube, a glass-calomel combination electrode, and a burette controlled by an automatic tit-

rator. Water (20 ml) and 10% palladium on carbon 65 catalyst (1.77 g) were added, and the system was flushed with nitrogen. Thereafter, a stream of hydrogen was bubbled through the suspension with stirring, a pH value of 2.5 being maintained in the aqueous phase by the addition of 0.5 N aqueous 70 hydrochloric acid via the automatic titrator. When the consumption of acid stopped, the flask was flushed with nitrogen until all hydrogen was removed, and the catalyst was filtered off. The aqueous phase was separated and freeze-dried to give 75 the desired compound as a colourless foam.

The NMR spectrum (D20) showed signals at 8 = 1.38 (s, 6H; 2-CH3), 1.46 (s, 3H; 2-CH3), 1.58 (s, 3H; 2-CHi), 3.56 (m, 2H; 6<x-H and 6/3-H), 4.60 (s, 1H; 3-H), 4.63 (s, 1H; 3-H), 5.03 (m, 1H; 5-H), 5.27 (s, 1H; 80 CW-NH2), 5.53 (s, 2H; 5-H and 6-H), 5.97 (bs, 1H; OCH2O), and 7.53 (s, 5H; arom. CH) ppm. Tetramethylsilane was used as external reference.

Example 2

1,1-Dioxopenicillanoyloxymethyl 6-[D- a-amino - a 85 - (p -hydroxyphenyl)acetamido\penicillanate, hydrochloride

A. 1,1 - Dioxopenicillanoyloxymethyl 6-[/V- (ben-zyloxycarbonyl)-D-a-amino -a-(p-hydrox-ypheny/)acetamido]penicillanate

90 To a solution of chioromethyl penicillanate 1,1-dioxide (1.41 g, 5 mmol)*' in dimethylformamide (25 ml) was added potassium 6 -[N - (benzyloxycar-bonyl) -D-a - amino -«- (p - hydroxyphenyl) -acetamido] penicillanate (2.46 g, 5 mmol), and the 95 mixture was stirred at room temperature for 18 hours. After dilution with ethyl acetate (100 ml), the mixture was washed with water (4 x 25 ml), dried, and evaporated/'n vacuo. The residual oil was purified by dry column chromatography on silica gel 100 (ethyl acetate-petroleum ether 8:2) to yield the desired compound as a yellowish oil.

B. 1,1 - Dioxopenicillanoyloxymethyl 6 -\D - a -amino -a- (p - hydroxyphenyljacetamidojpenicilla-nate hydrochloride

105 The benzyloxycarbonyl protecting group ofthe compound prepared in Example 2A was removed by hydrogenation at atmospheric pressure using the method described in Example 1B to afford the title compound as a colourless, amorphous product. 110 Example 3

1 -(1,1 - Dioxopenicillanoyloxyjethyl 6 - (D - a -amino -a- phenylacetamidojpenicillanate hydrochloride

By substituting a-chloroethyl 6 - (D - a - azido - a. -115 phenylacetamido)penicillanate forthe corresponding chioromethyl ester in the procedure of Example 1 A, 1 - (1,1 - dioxopenicillanoyloxyjethyl 6 - (D - a -azido - a - phenylacetamidojpenicillanate was obtained.

120 B. Following the procedure of Example 1 B, but substituting 1 -(1,1 -dioxopenicillanoyloxyjethyl 6-(D - a - azido -a- phenylacetamido)penicillanate for the 1,1 - dioxopenicillanoyloxymethyl 6-(D-a-azido- a- phenylacetamido)penicillanate, 1 - (1,1 -125 dioxopenicillanoyloxyjethyl 6 - (D - a - amino - a -phenylacetamidojpenicillanate, hydrochloride was orthe equivalent amount of the corresponding iodomethyl ester resulting in a much shorter reaction time.

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obtained as an amorphous product.

Example 4

1,1 -Dioxopenicil/anyloxymethyl6- (D,L - a -carboxy -a- phenylacetamidojpenicillanate sodium salt 5 A. 1,1- Dioxopenicillanoyloxymethyl 6 - (D,L - a -benzyloxycarbonyl- a-phenylacetamidojpenicillanate

Following the procedure described in Example 2

A, but substituting sodium 6 - (D,L - a - benzylox-

10 ycarbonyl - a- phenylacetamidojpenicillanate forthe potassium 6 -[N - benzyloxycarbonyl - D - a - amino -a- {p - hydroxyphenyl)acetamido] penicillanate, the desired compound was obtained.

B. 1,1 - Dioxopenicillanoyloxymethyl 6 -(D,L- a -

15 carboxy -a- phenylacetamidojpenicillanate sodium salt

To a solution of 1,1 - dioxopenicillanoyloxymethyl 6 - (D,L -a- benzyloxycarbonyl - a-phenylacetamido)penicillanate (1.43 g, 2 mmol) in 20 ethanol (20 ml) was added 10% palladium on carbon catalyst, and the mixture was hydrogenated at atmospheric pressure until the consumption of hydrogen ceased. The catalyst was removed by filtration, washed with ethanol, and the filtrate was 25 evaporated//? vacuo. The oily residue thus obtained was dissolved in ethyl acetate {15 ml), water (15 ml) was added, and the apparent pH in the aqueous phase was adjusted to 7.0 by addition of 0.2 N aqueous sodium hydroxide with stirring. The aqueous 30 phase was separated and freeze-dried to yield the desired compound as a yellowish foam.

Example 5

Clavulanoyloxymethyl 6-(D-a- amino -a-phenylacetamidojpeniciflanate hydrochloride 35 Byfollowing the procedure described in Example 1A, but substituting sodium clavulanate forthe potassium penicillanate 1,1-dioxide, clavulanoyloxymethyl 6 - D - a - azido - a -phenylacetamidojpenicillanate was obtained as a 40 yellowish oil.

By catalytic hydrogenation ofthe above intermediate according to the method described in Example 1B, the title compound was obtained as an amorphous powder. 45 Example 6

1,1 - Dioxo -6a- chloropenicillanoyloxymethyl 6 -(D -a- amino - a - phenylacetamidojpenicillanate hydrochloride

By following the method described in Example 2A, 50 but substituting chioromethyl 6a - chloropenicilla-nate 1,1-dioxide forthe chioromethyl penicillanate 1,1-dioxide and triethylammonium 6-[N - (1 - N,N -dimethylaminocarbonylpropen - 2-yi)-D-a - amino -a- phenylacetamidojpenicillanate forthe potas-55 sium 6 -[N - (benzyloxycarbonyl) - D - a - amino - a -{p - hydroxyphenyl) - acetamido] penicillanate, 1,1-dioxo-6a-chloropenicillanoyloxymethyl 6-[N-(1 -N,N - dimethylaminocarbonylpropen -2-yl)-D-a-amino -a- phenylacetamido] penicillanate was 60 obtained.

The protecting group in the above intermediate was removed by acid-catalyzed hydrolysis (pH ~ 3) ina 1:1 mixture of ethyl acetate and water to afford, after separation andfreeze-drying ofthe resulting 65 aqueous phase, the title compound as an amorphBNSDOCID: <GB 2044255A_I_>

ous product.

Example 7

-Bromopenici/lanoyloxymethy!6-(D- a-amino -a - phenylacetamidojpenicillanate hydrochloride 70 Byfollowing the method described in Example 2A, but substituting chioromethyl 6/3 - bromopenicillanate forthe chioromethyl penicillanate 1,1 - dioxide and triethylammonium 6-[N - (1 - N,N -dimethylaminocarbonylpropen - 2-yl)-D-a - amino 75 - a - phenylacetamidojpenicillanate forthe potassium 6 -[N - {benzyloxycarbonyl) - D - a - amino - a -(p - hydroxyphenyDacetamido] penicillanate, 6/3 -bromopenicillanoyloxymethyl 6 -[N - (1 - N,N -dimethylaminocarbonylpropen - 2-yl)-D-a - amino 80 -a-phenylacetamidojpenicillanatewasobtained.

The protecting group in the above intermediate " was removed by acid-catalyzed hydrolysis (pH ~ 3) in a 1:1 mixture of ethyl acetate and water to afford, after separation and freeze-drying ofthe resulting r 85 aqueous phase, the title compound as an amorphous product.

Example 8

1,1 - Dioxopenicillanoyloxymethyl 6-(D-a-amino -a - phenylacetamido j - penicillanate hydrochloride 90 A. Tetrabutylammonium 6-(D- a-amino -a-phenylacetamidoj-penicillanate

To a stirred, cooled (5°C) mixture of 6 - (D - a -amino - a - phenylacetamido)penicillanic acid trihy-drate (8.08 g) and tetrabutylammonium hydrogen 95 sulphate (6.9 g) in water (20 ml) and dich-

loromethane (40 ml) was added slowly 2 N aqueous sodium hydroxide (20 ml). The organic layer was separated, and the aqueous phase was extracted with dichloromethane (20 ml). The combined dich-100 loromethane layers were dried (MgS04) and evaporated/77 vacuo to leave a viscous oil. The oil was dissolved in ethyl acetate (100 ml), and residual dichloromethane was removed at reduced pressure.

After standing overnight at 5°C, the precipitated 105 crystals were collected, washed with ethyl acetate, and dried//? vacuo to give the title compound as colourless, silghtly hygroscopic crystals with melting point 125-130°C (decomp.). B. 1,1 -Dioxopenicillanoyloxymethyl 6-(D-a-110 amino -a- phenylacetamidojpenicillanate hydrochloride

To a stirred suspension of tetrabutylammonium 6 -(D - a - amino - a - phenylacetamidojpenicillanate (2.95 g) in ethyl acetate (20 ml) and dichloromethane 115 (5 ml) was added a solution of lodomethyl 1,1 -diox-openicillanate (1.9 g) in ethyl acetate (10 ml). After a -few minutes, an almost clear solution was obtained. Dichloromethane was removed at reduced pressure and precipitated tetrabutylammonium iodide was fil- * 120 tered off. From the filtrate the title compound was transferred to an aqueous phase (25 ml) with 1 N aqueous hydrochloric acid (pH 3.0,5°C), and from the aqueous phase backto an organic phase (ethyl acetate, 25 ml) with 0.5 M aqueous sodium hydrogen 125 carbonate (pH 7.0,5°C). The organic layer was washed with water and the desired compound was again transferred to an aqueous phase as described above. To the aqueous phase was added n-butanol, and the water was removed azeotropically by distil-130 lation in vacuo to give the title compound as colour

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less crystals, melting point 175-177°C (decomp.);[aj2°:+2010(c=1,H20)

Example 9

5 1,1 - Dioxopenicillanoyloxymethyl 6-(D-a- amino -a - phenylacetamidojpenicillanate

To a cold (5°C) solution ofthe compound prepared in Example 8 (631 mg) in water (10 ml), ethyl acetate (10 ml) was added, and the pH ofthe mixture was 10 adjusted to 7.0 by addition of 0.5 M aqueous sodium hydrogen carbonate with stirring. The organic layer was separated, washed with water, dried (MgS04), and evaporated//? vacuo to give the title compound as a colourless solid.

15 The IR spectrum (KBr) showed strong bands at 1780 and 1690 cm-1.

Example 10

1,1 - Dioxopenicillanoyloxymethyl 6 -\D- a- amino -20 a - (p- hydroxyphenyljacetamido\penicillanate hydrochloride

A. Tetrabutylammonium 6 - [D - a - amino - a-(p-hydroxyphenyljacetamido]penicillanate To stirred, cooled (5°C) solution of tetrabutylam-25 monium hydrogen sulphate (3.57 g, 10.5 mmol) in water (10 ml), a mixture of dichloromethane and n-butanol (9:1,20 ml) was added, followed by 2N sodium hydroxide to bring the pH to about 3. Amox-ycillin*' trihydrate 4.2 g, 10 mmol) was added, and 30 the pH adjusted to 9 with 2N sodium hydroxide. The organic layer was separated, and the aqueous phase was extracted twice with 10 ml portions of dichloromethane: n-butanol (9:1). The combined extracts were concentrated to a viscous oil in vacuo, 35 and the residue was dissolved in ethyl acetate (50 ml). Crystallization was induced by scratching, and, after standing at 5°C for 2 hours, the crystals were filtered off, washed and dried to give the title compound with a melting point of 148-151°C (decomp.). 40 B. 1,1 - Dioxopenicillanoyloxymethyl 6-\D - a -amino - a- (p -hydroxyphenyljacetamidojpenicilla-nate, hydrochloride

To a stirred, cooled (5°C) solution of tetrabutylammonium 6- [D-a-amino- a- (p- hydrox-45 yphenyl)acetamido]penicillanate (606 mg, 1 mmol) in acetonitrile (5 ml), iodomethyl 1,1 - dioxopenicil-lanate (373 mg, 1 mmol) dissolved in acetonitrile (2 ml) was added. After stirring for 10 minutes at 5°C, ethyl acetate (50 ml) was added, and the solvent was 50 stripped in vacuo. The residue was dissolved in ethyl acetate (20 ml), and crystallized tetrabutylammonium iodide was removed by filtration. To the filtrate, water (10 ml) was added, and the pH was adjusted to 3 with N hydrochloric acid. The aqueous 55 phase was separated and freeze-dried to give the title compound as a colourless powder.

The NMR spectrum [(CD3)2SO] showed signals at 8 = 1.37 (s,6H; 2-CW3), 1-50 (s, 6H; 2-CW3),3.46 (m,2H, 6a-H and 6/3-H), 4.46 (s, 1H; 3-H), 4.57 (s, 1H; 3-H), 60 5.04 (bs, 1H; C//NH2), 5.27 (m, 1H; 5-H), 5.58 (m, 2H; 5-H and 6-H), 5.96 (bs, 2H, 0CW20), 6.87 and 7.37 (2d, J=8.5Hz, 4H; arom. CH) ppm. TMS was used as internal reference.

*' 6-[D-a-Amino-a-(p-hydroxyphenyl)acetamido]penicillanic acid

Example 11

65 1 -(1,1 - Dioxopenicillanoyloxyjethyl 6-(D-a-amino - a - phenylacetamidojpenicillanate hydrochloride

To a solution of tetrabutylammonium 6- (D-a-amino -a- phenylacetamido)penicillanate (5.9g, 10 70 mmol) in dichloromethane (10 ml) and ethyl acetate (40 ml), 1-iodoethyl 1,1 - dioxopenicillanate (10.55 g of 40% purity, corresponding to 4.22 g, 10.9 mmol) dissolved in ethyl acetate (30 ml) was added. The clearsolution was immediately seeded with tet-75 rabutylammonium iodide, whereafter dichloromethane was removed//? vacuo, and separated tetrabutylammonium iodide was filtered off. From the filtrate, the title compound was transferred to an aqueous phase (50 ml) with N hydrochloric acid (pH 80 3.0,5°C) and from the aqueous phase to an organic phase (ethyl acetate, 50 ml) with sodium hydrogen carbonate (pH 7.0,5°C). The organic phase was washed with water, and the title compound was again transferred to an aqueous phase as described 85 above. Freeze-drying of the aqueous phase gave the title compound as a colourless powder.

The NMR spectrum (D20) showed signals at 8 = 1.38 (s, 6H; 2-CHz), 1.43 (s, 3H; 2-CW3), 1.55 (s,3H; 2-CH3), 1.56 (d, 3H; CHCW3), 3.50 (m, 2H; 6a-H and 90 6/3-//), 4.53 (s, 1H; 3-H), 4.55 and 4.59 (2s, 1H; 3-H), 4.96 (m, 1H; 5-H), 5.26 (s, 1H; C//NH2), 5.51 (s, 2H; 5-H and 6-H), 6.95 (m, 1H; CWCH3), and 7.51 (s, 5H; arom. CH) ppm.

95 Example 12

6p-Bromopenicillanoyloxymethyl6-(D- a-amino -a-phenylacetamidojpenicillanate hydrochloride

To a stirred solution of tetrabutylammonium 6- (D - a - amino - a - phenylacetamido)penicillanate (0.82 100 g, 1.4 mmol) in a mixture of ethyl acetate (2.8 ml) and dichloromethane (1.4 ml) was added a solution of iodomethyl 6/3-bromopenicillanate (0.60 g, 1.4 mmol) in ethyl acetate (5.6 ml). After stirring at room temperature for a few minutes, crystalline tet-105 rabutylammonium iodide began to precipitate. The dichloromethane was removed from the reaction mixture at reduced pressure, and the crystals were filtered off and washed with ethyl acetate (2 x 2.5 ml). The filtrate was washed with water (5 ml), to the 110 organic phase was added fresh water (10 ml), and the pH ofthe aqueous phase was adjusted to 3.1 by addition of 1 N hydrochloric acid with stirring. The aqueous phase was separated and freeze-dried to give the desired compound as a colourless foam. 115 The NMR spectrum (D20) showed signals at 8 = 1.34 (s, 3H; 2-CH3), 1.36 (s,3H; 2-Cf/3), 1.43 (s, 3H; 2-CHz), 1.58 (s, 3H; 2-CW3), 4.54 (s, 1H; 3-H), 4.75 (s, 1H; 3-H), 5.24 (s, 1H; CWNH2), 5.46-5.62 (m,4H; 5-H and 6-H), 5.88 (bs, 2H; OCHzO), and 7.47 (s, 5H; 120 arom. CH) ppm.

Example 13

1,1 - Dioxo -6a- chloropenicillanoyloxymethyl 6- (D -a- amino -a- phenylacetamidojpenicillanate hyd-125 rochloride

By substituting iodomethyl 1,1- dioxo - 6a -

BNSDOCID: <GB 2044255A_I_>

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chloropenicillanate forthe iodomethyl 6/3 -bromopenicillanate in the procedure of Example 12, the title compound was obtained as a colourless foam.

5 The NMR spectrum (D20) showed signals at S =

1.35 (s, 6H, 2-CH3), 1.41 (s,3H;2-0/3), 1.53 (s,3H; 2-CW3), 4.57 (s, 1H; 3-H), 4.73 (s, 1H; 3-H), 5.08 (s, 1H;

5-H or 6-H), 5.26 (s,1H; CWNH2), 5.34 (s, 1H; 5-H or

6-H), 5.49 (s, 2H; 5-H and 6-H), 5.94 (b, 2H; 0CW20), 10 and 7.49 (s, 5H; arom. CH) ppm.

Example 14

1,1 - Dioxopenicillanoyloxymethyl 6 -(D- a- amino -a - phenylacetamidojpenicillanate hydrochloride

A. 1,1 - Dioxo -6a- bromopenicillanoy/oxymethyi6 15 -(D-a- amino - a - phenylacetamidojpenicillanate hydrochloride

By substituting iodomethyl 1,1 -dioxo-6a-bromopenicillanate forthe iodomethyl 6/3 -bromopenicillanate in the procedure of Example 12, 20 the desired compound was obtained as a colourless foam.

The NMR spectrum (D20) showed signals atS =

1.36 (s, 6H; 2-CH3), 1.41 (s, 3H; 2-Ctf3), 1.54 (s, 3H; 2-QV3), 4.57 (s, 1H; 3-H), 4.71 (s, 1H; 3-H), 5.09 (s, 1H;

25 5-H or 6-H), 527 (s, 1H, CWNH2), 5.35 (s, 1H; 5-H or 6-H), 5.50 (s, 2H; 5-H and 6-H), 5.95 (b, 2H; 0CH20), and 7.50 (s, 5H; arom. CH) ppm.

B. 1,1 - Dioxopenicillanoyloxymethyl 6-(D-a-amino -a- phenylacetamidojpenicillanate hydroch-

30 loride

To a solution of 1,1 -dioxo - 6a - bromopenicil-lanoyloxymethyl 6 - (D - a - amino - a -phenylacetamido)penicil!anate (liberated from 1.36 g ofthe corresponding hydrochloride) in ethyl ace-35 tate (50 ml) was added water (25 ml) and 10% palladium on carbon catalyst (0.7 g), and the mixture was shaken in a hydrogen atmosphere for 40 minutes. After removal ofthe catalyst by filtration, the pH ofthe aqueous phase was adjusted to 2.5 40 with 1 N hydrochloric acid. From the separated aqueous phase the title compound was transferred to an organic phase (ethyl acetate, 25 ml) with aqueous potassium bicarbonate (pH 7.0,5°C) and back to a fresh aqueous phase with 1 N hydrochloric acid 45 (pH 2.7). The aqueous phase was freeze-dried to give the title compound as a colourless powder.

The NMR spectrum ofthe product was identical with that ofthe compound described in Example 1. Example 15

50 6/3-lodopenicillanoyloxymethyl6-(D-a-amino - a - phenylacetamidojpenicillanate hydrochloride

Following the procedure described in Example 12 but substituting iodomethyl 6/3-iodopenicillanate for the iodomethyl 6j3-bromopenicillanate, the title 55 compound was obtained as a colourless foam. The NMR spectrum (D20) showed signals at 8 = 1.33 (s, 3H; 2-CH3), 1.38 (s, 3H; 2-CH3), 1.45 (s, 3H; 2-CW3), 1.60 (s, 3H; 2-CH3), 4.56 (s, 1H; 3-H), 4.74 (s, 1H; 3-H), 5.22 (s, 1H; CWNH2), 5.3-5.7 (m, 4H; 5-H and 60 6-H), 5.92 (bs, 2H; 0CW20), and7.49 (s, 5H; arom. CH) ppm.

Example 16

6$- Chloropenicillanoyioxymethyl6-(D- a-amino -a - phenylacetamidojpenicillanate hydrochloride 65 Following the procedure described in Example 12,

but substituting iodomethyl 6/3-chloropenicillanate forthe iodomethyl 6/3-bromopenicillanate, the title compound was obtained as a colourless foam.

The IR spectrum (KBr) showed strong bands at 70 1790-1770 and 1690 cm"1.

Example 17 Clavuianoyloxymethyl 6 - (D - a - amino - a -phenylacetamidojpenicillanate hydrochloride A. lodomethyl6-(D-a-azido-a-75 phenylacetamidojpenicillanate

To a solution of chioromethyl 6 - (D - a - azido - a -phenylacetamido)penicillanate (1.32 g, 3 mmol) in acetone (25 ml), sodium iodide (1.80 g, 12 mmol) was added, and the mixture was stirred at room 80 temperature for 18 hours. The precipitate was filtered off, and the filtrate was evaporated in vacuo. The residue was extracted with ethyl acetate (25 ml), the extract was concentrated to about 3 ml and subjected to column chromatography on silica gel using 85 hexane ethyl acetate 1:1 as eluent. Fractions containing the desired compound were combined and evaporated/'/? vacuo to leave the title compound as a yellowish oil.

The NMR spectrum (CDCI3) showed signals at 8 = 90 1.58 (s,3H; 2-CH3), 1.67 (s,3H; 2-Ctf3),4.47 (s,1H; 3-H), 5.13 (s, 1H; CWN3), 5.52-5.82 (m, 2H; 5-H and 6-H), 6.00 (ABq, 2H; OCH2J), 7.4 (s, 5H; arom. CH), and 7.0-7.4 (m, 1H; CONW) ppm. TMS was used as internal reference.

95 B. Clavuianoyloxymethyl6- (D- a-azido -a-phenyiacetamidojpenicilianate

To a solution of iodomethyl 6 - (D - a - azido - a -phenylacetamidojpenicillanate (378 mg, 0.73 mmol) in hexamethyl phosphoric acid triamide (3.8 ml), 100 lithium clavulanate (90 mg, 0.44 mmol) was added, and the mixture was stirred at room temperature for one hour. The mixture was diluted with ethyl acetate (90 ml) and washed with water (3 x 20 ml) followed by saturated aqueous sodium chloride (10 ml), dried, 105 and evaporated//? vacuo. The yellow oil thus obtained was purified by column chromatography on silica gel using hexane-ethyl acetate 1:4 as eluent to yield the desired compound as a slightly yellowish foam.

110 The NMR spectrum (CDCQ showed signals at 8 = 1.51 (s,3H;2-Ctf3), 1.64 (s,3H; 2-CH3), 3.11 <d,J=17 Hz, 1H; 6-H), 3.51 (dd, J,=17 Hz, J2=3 Hz, 1H; 6-H), 4.25 (d, J=7 Hz, 2H; Ctf2OH); 4.51 (s, 1H; 3-H), 4.92 (m, 1H; =CH-), 5.13 (s, 1H; 5-H), 5,13 (s, 1H; 3-H), 115 5.5-5.8 (m, 3H; 5-H, 6-H, and CHN3), 5.89 (ABq, 2H; 0CW20), 7.16 (d, J=8.5 Hz, 1H; CONH), and7.41 (m, 5H; arom. CH) ppm. TMS was used as internal standard.

C. Clavuianoyloxymethyl 6-(D-a- amino - a -120 phenylacetamidojpenicillanate hydrochloride. A solution of clavuianoyloxymethyl 6 - (D - a -azido - a - phenylacetamidojpenicillanate (130 mg, 0.22 mmol) in ethyl acetate (20 ml) was placed in a three-necked flask, equipped with a gas inlet/outlet 125 tube, a glass-calomel combination electrode, and a burette. Water (20 ml) and 10% palladium/on/carbon catalyst (130 mg) were added, and the system was flushed with nitrogen. Hydrogen was passed through the stirred mixture, and the pH-value was 130 maintained at 2.5 by simultaneous addition of 0.1 N

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GB 2 044 255 A 13

aqueous hydrochloric acid. When the consumption of acid ceased, the flask was flushed with nitrogen, and the catalyst was filtered off. The aqueous layer was separated, filtered and freeze-dried to give the 5 desired compound as a colourless powder.

The NMR spectrum [(CD3)zS0] showed signals at 8 = 1.30 (s, 3H; 2-CHs), 1.44 (s, 3H; 2-CW3), 3,12 (d, J=17 Hz, 1H; 6-H), 3.65 {dd, J, = 17 Hz, J2=3 Hz, 1H; 6-H), 4.00 (m, 2H; C//2OH), 4.42 (s, 1H; 3-H), 4.75 (m, 10 1H; -CH=), 5.15 (bs, 1H; 3-H), 5.40-5.75 (m, 3H; 5-H, 6-H, and CtfNH2J, 5.85 (ABq, 2H; OCW2OJ, 7.50 (m, 5H; arom. CWJ, and 9.45 (d, J=7 Hz, 1H; CON//) ppm. TMS was used as internal reference.

Example 18

15 Clavuianoyloxymethyl 6 - [/? - a - amino - a-(p-hydroxyphenyljacetamido] - penicillanate hydrochloride

A. Chioromethyl 6-[N- benzyloxycarbonyl -D-a-amino - a-(p-hydroxyphenyljacetamido]penicifla-

20 nate

To a suspension of potassium 6 -[N - ben-zytocycarbonyl - D - a - amino - a - (p - hydrox-yphenyl)acetamido]penicillanate (2.46 g, 5 mmol) in N,N - dimethylformamide (25 ml) was added 25 chloroiodomethane (2.18 ml),30 mmol),andthe mixture was stirred at room temperature for 3 hours. After dilution with ethyl acetate (100 ml), the mixture was washed with water (4 x 25 mi), dried, and evaporated in vacuo. The residue was purified by 30 column chromatography on silica gel (using ethyl acetate/hexane 1:1 as eluent) to yield the desired compound as a yellowish oil.

B. Clavuianoyloxymethyl 6 - [D - a - amino -a-(p-hydroxyphenyljacetamido]penicilIanatehydroch-

35 loride

Byfollowing the procedure described in Example 17 A, 17 B, and 17 C but substituting chioromethyl 6 -[N - benzyloxycarbonyl - D - a - amino - a - (p -hydroxyphenyljacetamido] penicillanate for the 40 chioromethyl 6 - (D - a - azido - a -

phenylacetamidojpenicillanate, the title compound was obtained as a colourless freeze-dried powder.

The IR spectrum (KBr) showed strong bands at 1775 and 1690 cm"1. 45 Example 19

1,1 - Dioxopenicillanoyloxymethyl 6 - (D,L -a- carboxy -a- phenylacetamidojpenicillanate sodium salt A. 1,1 Dioxopenicillanoyloxymethyl 6 - (D,L - a -benzyloxycarbonyl - a- phenylacetamidojpenicilla-50 nate

To a suspension of sodium 6 - (D,L -a- benzyloxycarbonyl - a - phenylacetamidojpenicillanate (0.98 g,2 mmol) in N,N-dimethylformamide (10 ml), . iodomethyl penicillanate 1,1 - dioxide (0.75 g, 2 55 mmol) was added, and the mixture was stirred for30 min. at room temperature. Ethyl acetate (50 ml) was added, and the mixture was extracted with saturated aqueous calcium chloride (3x12 ml), dried, and evaporated//? vacuo. The oily residue was purified 60 by column chromatography on silica gel using hexane-ethyl acetate 1:1 as eluent to yield the desired compound as a yellowish oil.

The NMR spectrum (CDCI3) showed signals at 8 = 1.4-1.6 (m, 12H; 2-CW3),3.46 (m,2H; 6-H), 4.4-4.5 (m, 65 2H; 3-H and CtfCO), 4.56-4.65 (m, 2H; 3-H and 5-H),

5.19 (s, 2H; PhCtf20),5.4-5.75 (m, 2H; 5-H and 6-H), 5.9 (ABq, 2H; 0CW20), 7.3 (s, 5H; arom. CH), 7.35 (s, 5H; arom. CH), and 7.5-7.95 (m, 1H; CONW) ppm. TMS was used as internal reference. 70 B. 1,1 -Dioxopenicillanoyloxymethyl 6 - (D,L - a -carboxy -a- phenylacetamidojpenicillanate sodium salt

To a solution of 1,1 - dioxopenicillanoyloxymethyl 6 - (D,L - a - benzyloxycarbonyl - a -75 phenylacetamidojpenicillanate (1.0 g, 1.4 mmol) in ethyl acetate (25 ml), water (25 ml) and 10% palladium on carbon catalyst (1.0 g), were added, and the pH-value ofthe mixture was adjusted to 7.0. Hydrogen was bubbled through the stirred mixture, and 80 the pH-value was maintained at 7.0 by addition of 0.1 N aqueous sodium hydroxide. When the consumption of base stopped (after about 1 hour), the catalyst was filtered off, and the aqueous phase was separated, filtered, and freeze-dried to give the desired 85 compound as a colourless powder.

The NMR-spectrum (D20) showed signals at 8 = 1.47 (s, 3H; 2-CW3), 1.53 (s,3H; 2-CW3), 1.63 (s, 6H; 2-CH3), 3.55 (m, 2H; 6-H), 4,12 (s, 1H; 3-H), 4,17 (s, 1H; 3-H), 4.70 (s, 1H; WCO), 5.00 (m, 1H; 5-H), 90 5.4-5.7 (m, 2H; 5-H and 6-H), 6.00 (bs, 2H; OCHzO), 7.42 (s, 5H; arom. CH) ppm. TMS was used as external reference.

Example 20

1,1 - Dioxopenicillanoyloxymethyl 6/3 - aminopenicil-95 lanate hydrochloride

A. Tetrabutylammonium 6(3 - aminopenicillanate

To a stirred, ice-cooled mixture of 6/3-

aminopenicillanic acid (4.32 g, 20 mmol), tetrabutylammonium hydrogen sulphate (6.8 g, 20 100 mmol), dichloromethane (50 ml), and water (20 ml) was added slowly a solution of sodium hydroxide (1.60 g,40 mmol) in water (3.5 ml). The organic layer was separated, and the aqueous layer was extracted with dichloromethane (2x25 ml). The combined 105 organic layers were dried and evaporated//? vacuo to leave the desired compound as a viscous oil.

The IR spectrum (CHCI3) showed strong bands at 1760 and 1610 cm-1.

B. 1,1 - Dioxopenicillanoyloxymethyl 6(3 -110 aminopenicillanate hydrochloride

To a solution of tetrabutylammonium 6/3 -aminopenicillanate (5.1 g, 11 mmol) in ethyl acetate (25 ml) was added a solution of iodomethyl penicillanate 1,1 - dioxide (3.73 g, 10 mmol) in ethyl acetate 115 (25 ml). After stirring for 15 min. at room temperature, the precipitate was filtered off, and the filtrate was evaporated//? vacuo. The residue was purified by column chromatography on Sephadex ® LH 20 using chloroform-hexane 65:35 as eluent. The 120 purified product was dissolved in ethyl acetate (25 ml), water (25 ml) was added, and the pH-value of the mixture was adjusted to 2.0 by addition of 2 N hydrochloric acid.

The aqueous phase was separated and freeze-125 dried to give the title compound as a colourless powder.

The NMR spectrum (D20) showed signals at 8 = 1.52 (s, 3H; 2-CH3), 1.60 (s, 3H; 2-CH3), 1.65 (s, 3H; 2-CH3), 1.76 (s, 3H; 2-CH3), 3.52-3.8 (s, 2H; 6-H), 4.78 130 (s, 1H; 3-H), 4.90 (s, 1H; 3-H), 5.05-5.25 (m, 1H; 5-H),

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5.20 (d, J=4 Hz, 1H; 6-H), 5.78 (d, J=4Hz, 1H; 5-//), and 6.08 (bs, 2H; 0CW20) ppm. TMS was used as external reference.

Example 21

5 1,1 - Dioxopenicillanoyloxymethyl 6-(D-a-amino -a -phen ylacetamidojpenicillanate hydrochloride

To a stirred suspension of D - a - phenylglycyl chloride hydrochloride (1.98 g, 10 mmol) in dichloromethane (25 ml) was added at 0°C sodium car-

10 bonate (1.68 g, 20 mmol) followed by 1,1 - diox-openicillanoyioxymethyi 6 - aminopenicillanate hydrochloride (3.98 g, 8 mmol). After vigorous stirring at 0°Cfor 1.5 h,the mixture was evaporated in vacuo. The residue was taken up in an ice-cooled mixture of

15 ethyl acetate (25 ml) and saturated aqueous sodium hydrogen carbonate (25 ml). The organic phase was separated, water (20 ml) was added, and the pH-vatue ofthe mixture was adjusted to 2.5 by addition of 2N hydrochloric acid. The aqueous phase was

20 separated and freeze-dried to give an amorphous powder which crystallized from ethanol/butanone-2 to yield a product identical with that described in Example 8.

Example 22

25 1,1 - Dioxopenicillanoyloxymethyl 6-(D-a- amino -a - phenylacetamidojpenicillanate hydrochloride

A. Potassium 6 - [/V - (1 - dimethylaminocarbonylpropen -2-ylj-D - a-amino - a.-phenylacetamido\penicillanate

30 To a solution of triethylammonium 6 -[N - (1 -dimethylaminocarbonylpropen - 2-yl)-D-a - amino

- a - phenylacetamido] penicillanate (27.3 g, 48 mmol) in acetone (1 litre), 1 M potassium 2-ethylhexanoate in acetone (49 ml) was added drop-

35 wise. After stirring at room temperature for 2 hours, the precipitate was filtered off and recrystallized from methanol-isopropanol to afford the title compound; melting point: 201-203°C (dec.);[a]^0 = 174°

40 (c=1, water).

B. 1,1 - Dioxopenicillanoyloxymethyl 6-(D-a-amino - a-phenylacetamidojpenicillanate hydrochloride

To an ice-cooled solution of potassium 6 -[N - (1 -

45 dimethylaminocarbonylpropen- 2-yl)-D-a -amino

- a- phenylacetamidojpenicillanate (5.49 g, 11 mmol) in N,N - dimethylformamide (25 ml), iodomethyl penicillanate 1,1 - dioxide (3.73 g, 10 mmol) was added, and the mixture was stirred at 5°C

50 for 30 minutes. After dilution with ethyl acetate (100 ml), the mixture was extracted with water (4x25 ml) and saturated aqueous sodium chloride (25 ml). The organic phase was dried and evaporated in vacuo to half the initial volume. Water (25 ml) was added, and

55 the apparent pH-value ofthe mixture was adjusted to 2.5 by addition of 2N hydrochloric acid with stirring. During the hydrolysis this pH-value was maintained by addition of further hydrochloric acid. When the consumption of acid ceased (after about 30

60 minutes), the aqueous phase was separated and freeze-dried to give a compound, which after crystallization from ethanol/butanone-2 was identical with that described in Example 8.

Example 23

65 1,1 - Dioxo -6/3- (2,6 - dimethoxybenzamidojpenicil-

lanoyloxymethyl 6 - (D - a - amino - a -phenylacetamidojpenicillanate hydrochloride.

Sodium 6 - (D - a - amino - a -phenylacetamidojpenicillanate (0.75 g, 2 mmol) was 70 added to an ice-cold solution of iodomethyl

1,1-dioxo - 6/3 - (2,6 - dimethoxybenzamido)penicil-lanate (1.11 g, 2 mmol) in dimethylformamide (10 ml). The resulting solution was kept in an ice-bath for 30 minutes, diluted with ethyl acetate (40 ml) and 75 washed with water (4x10 ml). The organic phase was stirred with water while hydrochloric acid was added to pH 2.5. The aqueous phase was separated and freeze-dried to yield the title compound as a colourless powder.

80 The NMR spectrum (CD3OD, TMS as internal reference) showed signals at 8=1.47 (s, 3H; 2-CW3), 1.50 (s, 6H; 2-CH3), 1.58 (s, 3H; 2-Ctf3),3.83 <s,6H; OCW3), 4.50 (s, 1H; 3-H), 4.69 (s, 1H; 3-H), 5.18 (s, 1H;

CWNH2), 5.21 (d, J=4 Hz, 1H; 5-H), 5.4-58 (m, 2H; 5-H 85 and 6-H), 6.00 (m, 2H; 0CW20), 6.27 (d, J=4 Hz, 1H; 6-H), 6.73 (d, 2H; arom. 3-H and 5-H), 7.43 (t, 1H; arom. A-H), and 7.53 (s, 5H, arom. CH) ppm.

Example 24 1 -(1,1 - Dioxopenicillanoyloxyjethyl 6-[D-a-90 amino - a- (p -hydroxyphenyljacetamido\penicilla-nate hydrochloride

By following the procedure of Example 10B, but substituting 1 - iodoethyl 1,1 - dioxopenicillanatefor the iodomethyl 1,1 -dioxopenicillanate, the title com-95 pound was obtained as a colourless powder.

The IR spectrum (KBr) showed strong bands at 1785,1690, and 1655 cnrr1.

Example 25

613-Bromopenicillanoyloxymethyl6-\D-a-amino -100 a - (p-hydroxyphenyl)acetamido\penicillanate hydrochloride

By substituting iodomethyl 6/3-bromopenicillanate forthe iodomethyl 1,1 - dioxopenicillanate in the procedure of Example 10B, the title compound was 105 obtained as a slightly yellowish powder.

The IR spectrum (KBr) showed strong bands at 1790,1775, and 1690 cm"1.

Example 26

6/3 - lodopenicillanoyloxymethyl 6 - [O - a - amino - a 110 - (p - hydroxyphenyijacetamido]penicillanate hydrochloride

By following the procedure described in Example 10B, but substituting iodomethyl 6/3- iodopenicilla-nate forthe iodomethyl 1,1-dioxopenicillanate, the 115 title compound was obtained as an amorphous powder.

The IR-spectrum (KBr) showed strong bands at 1790,1775, and 1685 cm"1.

Claims (10)

120 1- A compound ofthe formula I:

Ri—CH—CO—MLj S/S\ /

125 " -c—0-CH-A

0 R3 J

in which R, stands for a phenyl, 4 - hydroxyphenyl, 1,4 - cyclohexadienyl or a 3 - thienyl group; R2 rep-130 resents a primary amino or a carboxy group; R3 is a

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hydrogen atom, or a lower alkyl, aryl or aralkyl radical, and A stands for a radical of a /8-iactamase inhibitor containing a 0-lactam ring as well as a carboxy group, A being connected via the carboxy 5 group, and salts ofthe compound of formula I with pharmaceutically acceptable, non-toxic acids or bases.

2. The pure diastereomers of the formula I of claim 1, mixtures thereof, as well as salts ofthe dias-

10 tereomers and mixtures thereof.

3. A compound of formula I of claim 1, in which R2 represents a primary amino group, and R, and A have the meanings defined in claim 1, and salts of the compound of formula 1 with pharmaceutically

15 acceptable, non-toxic acids.

4. A compound of formula I of claim 1, in which R2 represents a carboxy group, and Ri and A have the meanings defined in claim 1, and salts ofthe compound of formula I with pharmaceutically

20 acceptable, non-toxic bases.

5. A compound of formula I of claim 1, in which A is a radical selected from the group consisting of a) a radical ofthe formula II:

25

30

in which R4 stands for a hydrogen or a halogen atom; R5 is a hydrogen atom or an amino or 35 acylamino group, and where at least one of R4 and R5 is hydrogen;

b) a radical ofthe formula III:

40

R 8

R6Vt l/S

(T H X—0—

I 0

45 in which R6 stands fora halogen atom; and c) a radical ofthe formula IV:

50

E

55

in which R7 stands for a hydroxy group, or one ofthe radicals of known clavulanic acid derivatives with /3-lactamase inhibitory activity;

and salts thereof with pharmaceutically acceptable, 60 non-toxic acids or bases.

6. A compound of formula I and according to claim 5, in which A stands for a radical ofthe formula II, R4 and Rs both representing a hydrogen atom; and salts thereof as defined in claim 5. 65

7. A compound of formula I and according to claim 5, in which A stands for a radical of the formula III, R6 representing a halogen atom; and salts thereof as defined in claim 5.

8. A compound of formula I and according to

70 claim 7, in which R6 stands for bromine or iodine;

and salts thereof with pharmaceutically acceptable, non-toxic acids or bases.

9. A compound of formula I and according to claim 5, in which A stands for a radical of formula IV,

75 R7 representing a hydroxy group; and salts thereof as defined in claim 5.

10. 1,1- Dioxopenicillanoyloxymethyl 6 - (D - a -amino -a- phenylacetamidojpenicillanate, and salts thereof with pharmaceutically acceptable, non-toxic

80 acids.

11. 1 -{1,1 -Dioxopenicillanoyloxyjethyl 6-{D-a

- amino - a - phenylacetamido)penicillanate, and salts thereof with pharmaceutically acceptable, nontoxic acids.

85 12. 1,1-Dioxopenicillanoyloxymethyl 6-[D-a-amino - a - (p - hydroxyphenyljacetamido]penicillanate, and salts thereof with pharmaceutically acceptable, non-toxic acids.

13. 1,1 - Dioxopenicillanoyloxyethyl 6 -[D-a -

90 amino - (p - hydroxyphenyl)acetamido]penicillanate, and salts thereof with pharmaceutically acceptable, non-toxic acids.

14. 1,1 - Dioxo -6a - chloropenicillanoylox-ymethyl 6 - (D - a - amino - a -

95 phenylacetamidojpenicillanate, and salts thereof with pharmaceutically acceptable, non-toxic acids.

15. 1,1 - Dioxo - 6/3 - (2,6 - dimethoxyben-zamidojpenicillanoyloxymethyl 6 - (D - a-amino - a -phenylacetamidojpenicillanate, and salts thereof

100 with pharmaceutically acceptable, non-toxic acids.

16. 6/3-Bromopenicillanoyloxymethyl6-{D-a-amino - a - phenylacetamidojpenicillanate, and salts thereof with pharmaceutically acceptable, non-toxic acids.

105 17. 6/8-lodopenicillanoyloxymethyl 6-(D-a-amino - a - phenylacetamidojpenicillanate, and salts thereof with pharmaceutically acceptable, non-toxic acids.

18. 6/3-Bromopenicillanoyloxymethyl6-[D-a-

110 amino - a-(p- hydroxyphenyljacetamido]penicillanate, and salts thereof with pharmaceutically acceptable, non-toxic acids.

19. 6/3- lodopenicillanoyloxymethyl6 -[D - a-amino -a - (p - hydroxyphenyljacetamido]penicilla-

115 nate, and salts thereof with pharmaceutically acceptable, non-toxic acids.

20. 1,1 - Dioxopenicillanoyloxymethyl 6 - (D,L - a

- carboxy-a-phenylacetamidojpenicillanate, and salts thereof with pharmaceutically acceptable, non-

120 toxic bases.

21. Clavuianoyloxymethyl 6 - (D - a - amino - a -phenylacetamidojpenicillanate, and salts thereof with pharmaceutically acceptable non-toxic acids.

22. Clavuianoyloxymethyl 6 - [D - a - amino - a -

125 (p- hydroxyphenyljacetamido]penicillanate, and salts thereof with pharmaceutically acceptable, nontoxic acids.

23. A method for producing a compound of formula I of claim I, in which

130 a) a compound of formula V:

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in which R„ R3, and A are as defined above, B stands for an azido group, a protected amino group, or a 10 protected carboxy group, is subjected to a catalytic hydrogenolysis or hydrolysis depending on what A and B stand for; or b) a 6-aminopenicilfanic acid ester of formula XI:

or a trialkylsily! derivative thereof, in which R3 and A are as defined in claim 1, is reacted with a reactive derivative of an acid of formula XII:

25 R,-CH-COOH

L

in which R-, is as defined above, and B stands for NH34, Hal"; or

30 c) an aminopenicillin represented by the general formula XIII:

«ri"-t'-'dtssvx

Vr "-C—0—M 0 IE

40 is reacted with a compound of formula VIII:

A-CH-X

JL

45 in which formulae R„ R3, and A are as defined before, M stands for a cation, and X stands for a halogen atom or a similar leaving group,

whereafter the compound of formula I of claim 1 is recovered as such or in the form of a salt as defined 50 in claim 1.

24. The method of claim 23 a), in which B stands for a member selected from the group consisting of benzyloxycarbonylamino,triphenylmethylamino, 1 -methoxycarbonyl - propen - 2 - yl - amino, 1 - N,N -

55 dimethylaminocarbonylpropen - 2 - yl - amino, benzyloxycarbonyl, and cyanomethoxycarbonyl.

25. The method of claim 23 c), in which, in formula VIII, X stands for iodine.

26. A compound of formula VIII of claim 23, in 60 which A has the meanings defined in claim 5, and R3

represents hydrogen or methyl.

27. A compound according to claim 26, which is a halomethyl penicillanate 1,1-dioxide.

28. A compound according to claim 26, which is 65 an a-haloethyl penicillanate 1,1-dioxide.

29. A compound according to claim 26, which is a halomethyl clavulanate.

30. A compound according to claim 26, which is a halomethyl 6/3-bromopenicillanate.

70 31. A compound according to claim 26, which is a halomethyl 6/3-iodopenicillanate.

32. A pharmaceutical composition in dosage unit form for enteral parenteral ortopical treatment of patients (including animals) suffering from infecti-

75 ous diseases, which comprises as an active ingredient 0.025 g to 2.5 g of a compound as claimed in claim 1 together with an atoxic pharmaceutically acceptable carrier.

33. A pharmaceutical composition in dosage unit

80 form as claimed in claim 32 for oral treatment of patients, containing from 0.05 g to 1.5 g ofthe active ingredient.

34. A pharmaceutical composition in dosage unit form as claimed in claim 32 and containing as the ?

85 active component the compound 1,1- dioxopenicillanoyloxymethyl 6 - (D - a - amino - a -phenylacetamidojpenicillanate or a salt thereof with a pharmaceutically acceptable, non-toxic acid.

35. A pharmaceutical composition in dosage unit

90 form as claimed in claim 32 and containing as the active component the compound 1,1 - dioxopenicillanoyloxymethyl 6 - [D - a - amino - a - (p-hydroxyphenyDacetamido]penicillanate or a salt thereof with a pharmaceutically acceptable, non-

95 toxic acid.

36. A pharmaceutical composition in dosage unit form as claimed in claim 32 and containing as the active component the compound 6/3 - bromopenicil-lanoyloxymethyl 6 - (D - a - amino - a -

100 phenylacetamidojpenicillanate or a salt thereof with a pharmaceutically acceptable, non-toxic acid.

37. A pharmaceutical composition in dosage unit form as claimed in claim 32 and containing as the active component the compound 6/3- iodopenicil-

105 lanoyloxymethyl 6 - (D - a - amino - a -

phenylacetamidojpenicillanate ora salt thereof with a pharmaceutically acceptable, non-toxic acid.

38. A pharmaceutical composition in dosage unit form as claimed in claims 34,35,36, or 37 in the form

110 of tablets, pills, or capsules.

39. A pharmaceutical composition containing a compound as claimed in claim 1 together with carrier substances and auxiliary agents, containing from 1% to 95% of the active compound.

115 40. A compounded pharmaceutical composition as claimed in claim 39 containing the active ingredient together with a known amidinopenicillanic acid derivative, the ratio between the active compounds being between 1:20 and 20:1, preferably between

120 1:5 and 5:1.

41. A compounded pharmaceutical composition as claimed in claim 40, in which the active ingredient is 1,1-dioxopenicillanoyloxymethyl 6- (D - a - amino - a - phenylacetamidojpenicillanate and the

125 amidinopencillanic acid derivative is pivaloyloxymethyl 6 - [(hexahydro -1H - azepin -1 - yl) -methyleneamino] - penicillanate, the above active compounds optionally being used in the form of salts with a pharmaceutically acceptable, non-toxic

130 acid.

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42. In the treatment of non-human patients suffering from infectious diseases, the administration of a compound of formula I of claim 1 in an amount of 3-200 mg/kg body weight ofthe patient/day, or an

5 equivalent amount of a salt, as defined in claim 1, of a compound of formula I.

43. A compound of formula I defined in claim 1 substantially as hereinbefore described in any one of the foregoing examples.

10 44. A method of producing a compound of formula I as defined in claim 1 substantially as hereinbefore described in any one ofthe foregoing examples.

Printed for Her Majesty's Stationery Office by The Tweeddale Press Ltd., Berwick-upon-Tweed, 1980.

Published atthe Patent Office, 25 Southampton Buildings, Londonr WC2A1 AY, from which copies may be obtained.

BNSDOCID: <GB 2044255A__L>